Drugs which target DNA through direct crosslinking or trapping of topoisomerase I (TOP1) complexes are some of the most effective treatments for cancer. A more complete understanding of the molecular pathways that influence how cancer cells cope with these lesions may identify new targets for drug development with the goal of increasing therapeutic effectiveness through combination chemotherapy. The E3 ubiquitin ligase Rad18 plays a crucial role in postreplication repair (PPR) by monoubiquitinating PCNA in response to stalled replication forks. This event promotes lesion bypass by recruiting a group of specialized DNA polymerases that copy DNA templates containing DNA lesions through a process termed Translesion DNA synthesis (TLS). TLS is believed to be important for replicative bypass of cisplatin adducts and may be an important mechanism by which tumor cells resist therapy. To further probe into how TLS may impact chemotherapy, we used siRNA to deplete human cells of Rad18, Rev3 (the catalytic subunit of Pol ?), or two additional TLS polymerases, Rev1 and DNA Polymerase Eta, all four believed to be involved in replicating DNA containing cisplatin adducts. We found that all four gene products were necessary to prevent replication fork stalling following exposure to cisplatin consistent with a model that lesion bypass is accomplished by PCNA monoubiquitination and coordination of multiple TLS polymerases. Unexpectedly, we found that Rad18 and Rev3-depleted cells are hypersensitive to cisplatin suggesting they play additional roles in tolerance to cisplatin. We also observed that Rad18 colocalizes with DNA double strand breaks (DSBs) when cells are exposed to ionizing radiation, camptothecin, Mitomycin C, or cisplatin and that depletion of Rev3 leads to cellular phenotypes consistent with cells deficient in DSB repair. We hypothesize that Rad18 and Pol 6 possess alternative functions in addition to performing PPR. We believe these functions are important for resolution of replication-associated DSBs, a common intermediate during repair of ICLs and replication associated DSBs caused by TOP1 inhibitors. To address this hypothesis, we propose the following three specific aims: 1. Determine whether Rad18 or Pol ? promotes cell survival and prevents accumulation of chromosomal aberrations when cells are challenged with agents that induce replication-associated DSBs. 2. Determine whether Rad18 or Pol ? is necessary for the efficient repair of replication-associated DSB and whether Rad18 or Pol 6 facilitates HR triggered by camptothecin and ICL-inducing agents by measuring HR activity in cells and frequencies of drug-induced sister chromatid exchanges at the chromosomal level. 3. Determine the functional significance of localization of Rad18 to sites of DSBs by examining whether localization is dependent upon DNA replication and whether PCNA ubiquitination is associated with this event. We will also characterize the functional domains within Rad18 that are necessary for localization and whether those domains are important for cellular resistance to replication-associated DSBs and directing DNA repair. PUBLIC HEALTH RELEVANCE: Agents which target Topoisomerase I and introduce interstrand DNA crosslinks are some of the most widely used drugs used to treat cancer. We hypothesize that two enzymes, Rad18 and DNA Polymerase Zeta, are important for facilitating repair of DNA double stranded breaks associated with the therapeutic effectiveness of these agents. The studies proposed here will advance our understanding of DNA repair activities that modulate the sensitivity of cancer cells to these anticancer agents and provide new insight into how cells prevent genomic instability, a common hallmark of cancer.